Reverse transcription reactions are classified into reactions employing random primers, and reactions employing target-specific primers. In reverse transcription reactions for detection of a specific target, such as diagnostic kits for detection of RNA virus or the like, target-specific primers are generally used because they show higher sensitivity. In such reverse transcription reactions, specificity and sensitivity are determined by the high selectivity of primers that bind specifically to a target RNA sequence. However, because all components required for a reverse transcription reaction are mixed at room temperature, a non-specific reverse transcription reaction is caused by non-specific priming under this condition. Even at room temperature, non-specific priming occurs due to the high activity of reverse transcriptase to produce a number of unspecific cDNAs, and thus it is a major factor that increases non-specific amplification reactions. Due to such non-specific reverse transcription reactions, primers required for subsequent PCR reactions and limited concentrations of other essential components are consumed, and for this reason, the non-specific reverse transcription reactions act as competitive inhibitors. Non-specific reverse chain reactions are problematic when detecting a low concentration of RNA, and particularly, interfere with detection of a target RNA that is present in a very small amount in a solution that contains large amounts of RNAs extracted from cells or body fluids and having a high nucleotide sequence complexity. Thus, non-specific reverse transcription reactions make it difficult to detect a virus or gene present at a low concentration. Also, in multiplex reverse transcription reactions that are performed using various primers at the same time, non-specific reverse transcription reactions reduce specificity, making multiple detections difficult. This non-specific amplification is more greatly influenced by the relative amounts of the target nucleic acid and other nucleic acids derived from a biological sample than the absolute amount of the target nucleic acid. This is because non-specific hybridization of primers increases to increase non-specific reactions, when many RNAs other than a desired target are present in a reaction mixture.
In order to reduce such undesirable non-specific reactions and amplification, various attempts have been made. Specifically, there was an attempt to increase the detection limit of a specific target by more strongly hybridizing primers to the target. For example, a method was reported, in which the 5′ terminus of primers are substituted with LNA so that it can more strongly hybridize to the target, thereby reducing non-specific amplification (Malgoyre A. et al., Biochem Biophys Res Commun. Mar. 2, 2007; 354(1):246-52). As another method for solving problems using a specific primer structure, a method of preventing primer dimer formation was also developed. When a reverse transcription reaction is performed particularly at low temperatures, polymerization can proceed by partial hybridization between primers to form a dimer, an important factor that rapidly reduces sensitivity in the reverse transcription reaction. In order to solve this problem, it was proposed to use primers formed by extending complementary nucleotide sequences so that five nucleotides of the 5′ nucleotide sequence of the primers can form a hairpin structure at low temperature (Ji Young Hong et al., Virol J. 2011; 8: 330. Published online 2011; Korean Patent No. 10-0987352). However, this lock primer method has disadvantages in that, because the primers have high hybridization temperature due to the nucleotide sequence added to the 5′ terminus, non-specific hybridization of the primers to non-specific targets having nucleotide sequences similar thereto in a subsequent PCR reaction can be induced, and the efficiency of hybridization in the hybridization step is reduced due to the hairpin structure of the primers.
To solve such problems, a method was developed which uses blocked primers that are partially complementary to primers and that are blocked at the 3′ terminus. Such blocked primers have advantages in that, because they are blocked at the 3′ terminus, they do not act as primers in a nucleic acid polymerization reaction, and because they are short in length, they hybridize to primers only at room temperature to prevent primer dimer formation, and because they are detached immediately from the primers and are not operated when the temperature increases in a subsequent reaction, instant reverse transcription PCR can be performed (Korean Patent Application No. 10-2011-0017226).
In addition, a method of performing a reverse transcription reaction at high temperature for more specific hybridization to a target RNA was developed. It was reported that, when a cDNA is synthesized at high temperature such as 70° C. using primers that hybridize at high temperature and a reverse transcription polymerase that operates even at high temperature, it can be more specifically amplified (Fuchs B, et al., Mol Biotechnol, 1999 October; 12(3):237-40).
In multiplex reverse transcription PCRs, specificity is more important. This is because several primers are used in the reaction, a non-specific reaction can occur in a subsequent PCR reaction due to a non-specific reaction in the reverse transcription reaction step. To avoid this non-specific reaction in multiplex reverse transcription PCRs, a reverse transcription reaction is first performed, and then a PCR reaction is performed, so that a more specific reaction can be achieved. However, in this case, the process of opening a reaction tube and adding a reaction solution to the open reaction tube is troublesome, and the possibility of contamination in the process of opening and operating the reaction tube is higher. For this reason, it is generally preferable to sequentially perform a reverse transcription reaction and PCR in a single tube. Thus, a method of physically separating reactions from each other while performing reverse transcription PCR in a single tube without opening the tube was developed, in which the reverse transcription reaction mixture is present at the bottom of the tube during the reaction, and the PCR reaction mixture is suspended from the cover. In this method, the reverse transcription reaction is performed in the tube while the PCR reaction mixture is suspended from the cover and is not reacted, and after completion of the reverse transcription reaction, and the reaction tube is rotated in a centrifuge so that the PCR solution suspended from the cover of the reaction tube is mixed with the reverse transcription reaction product, after which the PCR reaction is performed (Rodney Mark Ratcliff, et al., 2002 November; 40(11): 40914099. doi: 10.1128/JCM.40.11.4091-4099.2002).
As a result of efforts to solve the above-described problems occurring in conventional reverse transcription reactions, a “hot-start reverse transcription reaction” was developed. The hot-start reverse transcription reaction is a method for detecting a very small amount of a target RNA, in which a reverse transcription reaction can be initiated at a high temperature at which priming could occur only to an RNA having a nucleotide sequence exactly complementary to primers, thereby preventing non-specific priming from occurring at room temperature and preventing non-specific primer oligomerization, thereby increasing the specificity of the reverse transcription reaction. To implement this method, a method that uses a heat-resistant polymerase and an aptamer was developed and is being used. A light cycler RNA master kit (Roche) is a method employing Tth DNA polymerase and an aptamer. Tth DNA polymerase combines a function of polymerizing DNA using RNA as a template and a function of polymerizing DNA using DNA as a template. The aptamer used herein is attached to the reactive site of Tth DNA polymerase, and thus is inactive at room temperature. When the temperature of the reaction solution is increased to high temperature, the three-dimensional structure of the aptamer is modified so that it is separated from Tth DNA polymerase so as to be active, and the reverse transcription reaction of a specifically primed target RNA can be performed, and afterwards PCR can be performed. In addition, the GeneAmp AccuRT Hot Start RNA PCR kit (Applied Biosystems) uses a heat-resistant polymerase derived from Thermus specie Z05 and can perform a reverse transcription reaction and PCR by using a single enzyme, like the Tth DNA polymerase. Also, the reaction is performed using an aptamer specific thereto. Such products can reduce non-specific amplification in reverse transcription PCR by inhibiting enzymatic activity at room temperature using the aptamer to reduce non-specific reverse transcription reactions. However, when the temperature of the aptamer is increased to high temperature, the three-dimensional structure thereof is modified, but when the temperature is lowered, the aptamer is restored to its original structure to inhibit DNA polymerases. In other words, the aptamer has the problem of reversibly inhibiting DNA polymerases. For the GeneAmp AccuRT Hot Start RNA PCR kit, it is described that the aptamer is still attached to DNA polymerase even at 55° C. and is completely detached when the temperature becomes 65° C. Thus, in reactions in which the annealing temperature of primers that are generally used is 55° C. or below, there is a problem in that the activity of DNA polymerase is inhibited to reduce PCR efficiency. For this reason, a heat-resistant DNA polymerase such as Tth DNA polymerase, which has reverse transcription function, is used. This heat-resistant DNA polymerase has a problem in that, because it has reverse transcription function during a subsequent PCR process in which enzymatic activity is maintained, it causes a continuous non-specific reverse transcription reaction, resulting in non-specific amplification.
A hot-start PCR reaction method employing antibodies is applicable only to Taq DNA polymerase (Enneth, G. et al., 1994, Biotechnology, 12; 506-509). In order to perform hot-start reverse transcription reactions using other kinds of reverse transcription polymerases, a heat-resistant reverse transcriptase that is resistant at high temperature should be used, and for reverse transcription PCR, a reverse transcriptase-specific antibody that is detached at low temperature and a DNA polymerase-specific antibody that is detached at high temperature are required. For this reason, antibodies specific to the respective heat-resistant polymerases should be developed, and thus there is a technical limit to such hot-start reverse transcription reactions.
The present inventors developed a hot-start PCR method that uses pyrophosphate (PPi) and heat-resistant pyrophosphatase (PPase) (Korean Patent No. 10-0292883). This method is based on the principle in which PPi that strongly binds to magnesium ions essential for DNA polymerization is added to inhibit a polymerase reaction at room temperature, and then PPase is reacted at high temperature to remove PPi. Reverse transcriptase cannot be used in a hot-start method that uses an antibody, because of its low activation temperature. A conventional hot-start PCR method that uses an antibody is a method in which the activity of enzyme is inhibited by enzyme-antibody binding at low temperature, and the antibody loses its binding with the enzyme due to a decrease in its stability when it reaches high temperature, whereby a PCR reaction occurs. Reverse transcriptase has no thermal stability at high temperature, unlike DNA polymerase. When the antibody reaches high temperature to lose its binding with reverse transcriptase, the activity of reverse transcriptase is inhibited. In an attempt to solve this problem, a hot-start reverse transcription reaction method that uses PPi and PPase has advantages in that it can be generally applied regardless of the kind of DNA polymerase and in that reactivity can be continuously maintained by continuously removing PPi generated from dNTP during PCR. However, if a hot-start PCR master mix solution is made using this method, PPase will slowly dissolve PPi from Mg2+ ions so that the activity of DNA polymerase will appear, and ultimately, a desired hot-start PCR reaction effect will be lost. In addition, if a reverse transcription reaction master mix solution containing PPase is made, it has a problem in that the activity of PPase at room temperature or 4° C. is maintained only for a short period of time, because PPase is very unstable. Thus, a mixture in a dried form having increased stability was developed (Korean Patent No. 10-1098764). Accordingly, in order to solve the above-described problems of hot-start reverse transcription reactions, technology for more highly sensitive hot-start reverse transcription and reverse transcription PCR is required.